The goal of this research program is to define mechanisms that promote mammalian oogenesis in vivo using model systems for oocyte development in vitro. Mouse oocytes, including those from primordial follicles, can grow in vitro and acquire competence to undergo embryogenesis. However, their growth and development in vitro does not produce oocytes equivalent to those grown in vivo. Furthermore, there may be long-term health consequences of aberrant oocyte development in vitro: Experiments will (1) address the nature of defects in oocytes grown in vitro and their consequences to subsequent embryogenesis and long-term health of offspring and (2) explore the communication between oocytes and granulosa cells and its impact on oocyte development. Specific Aim 1 will determine whether defects in oocyte development in vitro are due to failures in nuclear and/or cytoplasmic processes. Female pronuclei, male pronuclei, or both pronuclei will be microsurgically exchanged between fertilized eggs derived from oocytes grown in vitro and in vivo. Developmental potential and epigenetic programming in nuclear transplantembryos will be assessed. Specific Aim 2 will determine whether conditions of oocyte development in vitro affect post-implantation development and long-term health of offspring. Genetically marked embryos derived from in vitro-grown and in vivo-grown oocytes will be transferred together to recipient mothers. Offspring will undergo a battery of health-evaluating protocols such as gross assessment, blood chemistry, hematology, and histopathologicalevaluation.This will be the first systematic assessment of both short- and long-term health effects of oocyte development in vitro. Specific Aim 3 tests the hypothesis that a communication loop between oocytes and oocyte-associated granulosa cells (OAGCs) regulates the differentiation of OAGCs in ways that promote normal oocyte development. Paracrine factors secreted by oocytes affect the differentiation and function of OAGCs differently depending upon the stage of oocyte development. During oocyte growth in vitro, appropriate differentiation of OAGCs in cultured oocyte-granulosa cell complexes will be either augmented or perturbed by co-culture of complexes with denuded oocytes that either match or mismatch the developmental stage of the oocyte resident in the complex. Effects on growth, maturation, fertilization, epigenetic programming, and developmental competence of granulosa cell-enclosed oocytes will be determined. Taken together, these studies will advance understandingof the mechanisms of oogenesis, improve conditions for oocyte development in vitro, and define the consequences of suboptimal conditions during oogenesis.